Myron Christodoulides

Immunization with recombinant class 1 outer-membrane protein from Neisseria meningitidis: influence of liposomes and adjuvants on antibody avidity, recognition of native protein and the induction of a bactericidal immune response against meningococci.

The porA gene from Neisseria meningitidis was cloned into the pRSETA vector and recombinant class 1 outer-membrane protein expressed at high levels in Escherichia coli. The protein was readily purified by affinity chromatography on a Ni2+ matrix and used for immunization of mice with conventional AI(OH)3 adjuvant, with experimental adjuvants which have the potential for human use, and with liposomes. The resulting sera were analysed for the magnitude, subclass distribution and antigenic specificity of the immune response. In addition, surface plasmon resonance (SPR) was used to quantify antibody avidity by analysis of the kinetics of binding to native class 1 protein. Immunization with conventional and experimental adjuvants induced antibodies of low avidity that did not recognize native class 1 protein. In contrast, immunization with recombinant protein in liposomes induced antibodies of high avidity which recognized native class 1 protein, as measured by their ability to label meningococcal cells in immunofluorescence assays and to inhibit the binding of a protective mAb. These properties were associated with the presence in sera of high levels of antibodies with the ability to induce complement-mediated killing of meningococci. These data show that liposomes containing recombinant class 1 protein represent a potential basis of future vaccines, of defined composition, designed for the prevention of group B meningococcal infections.

Dynamics of Carriage of Neisseria meningitidis in a Group of Military Recruits: Subtype Stability and Specificity of the Immune Response following Colonization

Meningococcal carriage and the immune response to colonization were studied in a group of military recruits undergoing basic training. Subtyping by determination of the class 1 protein sequence clearly differentiated between strains and demonstrated the dynamics of carriage and transmission. Expression of class 1 protein by each strain remained stable during prolonged carriage by different subjects. Following colonization, a marked increase in serum bactericidal response occurred, which was specific for the subtype of the acquired strain and was associated with an increase in reactivity by Western blot to the homologous class 1 protein. Subjects colonized by multiple strains showed evidence of a specific immune response to the class 1 protein of each strain acquired. The subtype specificity of the bactericidal response to meningococci and the stability of expression of the class 1 protein have important implications for the design of vaccines for prevention of serogroup B meningococcal disease.

Proteomic Analysis of Outer Membranes and Vesicles from Wild-Type Serogroup B Neisseria meningitidis and a Lipopolysaccharide-Deficient Mutant

ABSTRACT Current experimental vaccines against serogroup B Neisseria meningitidis are based on meningococcal outer membrane (OM) proteins present in outer membrane vesicles (OMV) in which toxic lipopolysaccharide is depleted by detergent extraction. Knowledge of the composition of OM and OMV is essential for developing new meningococcal vaccines based on defined antigens. In the current study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nanocapillary liquid chromatography-tandem mass spectrometry were used to investigate the proteomes of OM and OMV from meningococcal strain MC58 and OM from a lipopolysaccharide-deficient mutant. The analysis of OM revealed a composition that was much more complex than the composition that has been reported previously; a total of 236 proteins were identified, only 6.4% of which were predicted to be located in the outer membrane. The most abundant proteins included not only the well-established major OM proteins (PorA, PorB, Opc, Rmp, and Opa) but also other proteins, such as pilus-associated protein Q (PilQ) and a putative macrophage infectivity protein. All of these proteins were also present in OMV obtained by extraction of the OM with deoxycholate. There were markedly increased levels of some additional proteins in OM from the lipopolysaccharide-deficient mutant, including enzymes that contribute to the tricarboxylic acid cycle. In all the preparations, the proteins not predicted to have an OM location were predominantly periplasmic or cytoplasmic or had an unknown location, and relatively few cytoplasmic membrane proteins were detected. However, several proteins that have previously been identified as potential vaccine candidates were not detected in either OM preparations or in OMV. These results have important implications for the development and use of vaccines based on outer membrane proteins.

Immunization with the Recombinant PorB Outer Membrane Protein Induces a Bactericidal Immune Response against Neisseria meningitidis

ABSTRACT Infections with Neisseria meningitidis are characterized by life-threatening meningitis and septicemia. The meningococcal porin proteins from serogroup B meningococci have been identified as candidates for inclusion in vaccines to prevent such infections. In this study, we investigated the vaccine potential of the PorB porin protein free of other meningococcal components. The porB gene from a strain of Neisseria meningitidis expressing the class 3 outer membrane porin protein (PorB3) was cloned into the pRSETB vector, and the protein was expressed at high levels in a heterologous host Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and used for immunization after incorporation into liposomes and into micelles composed either of zwitterionic detergent or nondetergent sulfobetaine. The immunogenicity of these preparations was compared to recombinant PorB protein adsorbed to Al(OH)3 adjuvant as a control. Although sera raised against the protein adsorbed to Al(OH)3 reacted with the purified recombinant protein, sera raised against liposomes and micelles showed greater activity with native protein, as measured by enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Reactivity with native protein was considerably enhanced by incorporation of the adjuvant monophosphoryl lipid A into the liposome or micelle preparations. Recognition of the native protein was in a serotype-specific manner and was associated with the ability of the antisera to promote high levels of serotype-specific complement-mediated killing of meningococci. These results demonstrate that the PorB protein should be considered as a component of a vaccine designed to prevent serogroup B meningococcal infection.

Interactions of Neisseria meningitidis with cells of the human meninges

The interaction of Neisseria meningitidis with the meninges that surround and protect the brain is a pivotal event in the progression of bacterial meningitis. Two models of the human meninges were established in vitro, using (i) sections of fresh human brain and (ii) cultures of viable cells grown from human meningiomas. Neisseria meningitidis showed a specific predilection for binding to the leptomeninges and meningeal blood vessels in human brain and not to the cerebral cortex. There was a close correlation between the adherence of different Neisseria species to leptomeninges and cultured cells. The major ligand that mediated adherence was the pilus, and pilin variation modulated the interactions. The presence of Opa protein increased the association of Cap+ meningococci that expressed low‐adhesive pili, but did not influence the association of high‐adhesive pili. In contrast, Opc did not influence the adherence of Cap+ meningococci, whereas loss of capsule was associated with a more intimate interaction between the bacteria and the meningioma cell that was not apparent with Cap+ meningococci. There was no evidence of internalization of meningococci by meningioma cells in vitro, an observation that is consistent with the barrier properties of the leptomeninges to N. meningitidis observed in vivo.

Declining serotype coverage of new pneumococcal conjugate vaccines relating to the carriage of Streptococcus pneumoniae in young children

BACKGROUND Asymptomatic carriage of the opportunistic pathogen Streptococcus pneumoniae is known to precede the development of invasive disease. Young children are one of the major reservoirs for pneumococci and worldwide over 700,000 children under two years old die due to invasive pneumococcal disease each year. Heptavalent conjugate vaccine (PCV-7) was introduced into the UK childhood immunisation schedule in September 2006. Our objective was to assess the emergence of colonising serotypes in young children in the three years following PCV-7 implementation. METHODS Time-series prevalence survey set in the paediatric outpatients department of a large UK teaching hospital. Participants were children aged four years and under attending the outpatients department during PCV-7 introduction (October 2006-February 2007) and in the same months of the two subsequent years. The main outcome measure was prevalence of pneumococcal carriage by serotype. RESULTS The rate of pneumococcal nasopharyngeal carriage remained stable during the three year period. We observed a significant 69% (95% CI, -40% to -118%, p<0.0001) decrease in carriage of PCV-7 serotypes during PCV-7 implementation and a concomitant increase in the proportion of non PCV-7 serotypes. The most prevalent emerging non-vaccine serotypes were 6C, 11A, 19A and 22F. By March 2009, PCV-13 was predicted to cover only 33.3% (95% CI, 24.2-42.5%) of strains carried in the study population. CONCLUSIONS Although the overall pneumococcal carriage rate remained stable between 2006 and 2009, we observed a significant decrease in the serotype coverage of PCV-7 and PCV-13. PCV-7 was highly successful in reducing carriage of vaccine serotypes. However, the increase in the proportion of non-vaccine serotypes found both in our study and causing invasive disease currently in the UK, underlines the importance of continued surveillance of carriage and disease.

Immunization with synthetic peptides containing epitopes of the class 1 outer-membrane protein of Neisseria meningitidis: production of bactericidal antibodies on immunization with a cyclic peptide

The class 1 outer-membrane protein of Neisseria meningitidis is the target for subtype-specific, bactericidal monoclonal antibodies (mAbs). The epitopes recognized by these antibodies have been mapped previously to linear peptides corresponding to the sequences thought to be exposed at the apices of surface-exposed loops of the protein. In this work several synthetic peptides containing the subtype Pl.16b epitope have been synthetized with the aim of inducing a polyclonal immune response resembling the reactivity of the mAbs. Initially, peptides of 9 and 15 amino acid residues were synthesized and used for immunization after coupling to a carrier protein. The reactivity of the resulting antisera, with synthetic linear decapeptides, resembled that seen in previous epitope mapping experiments with the protective mAbs. However, despite the induction of antibodies having the desired specificity, the antisera reacted poorly with the native protein in outer membranes, and were non-bactericidal. A 36mer peptide, consisting of the entire surface-exposed loop 4 of the class 1 protein was then synthesized and used for immunization as (i) free peptide, (ii) peptide coupled to carrier and (iii) peptide subjected to cyclization, in an attempt to restrict it to conformations that might more closely resemble the native loop structure. In contrast to antisera raised against linear peptides, antibodies raised by immunization with the 36mer cyclic peptide, did not react with linear peptides recognized by the mAbs, but instead appeared to recognize conformational determinants. This antiserum promoted complement-mediated bactericidal killing of the homologous meningococcal strain, demonstrating the potential of synthetic peptide immunogens for inducing a protective immune response against group B meningococci.

Interaction of primary human endometrial cells with Neisseria gonorrhoeae expressing green fluorescent protein

Infection of the endometrium by Neisseria gonorrhoeae is a pivotal stage in the development of pelvic inflammatory disease in women. An ex vivo model of cultures of primary human endometrial cells was developed to study gonococcal–host cell interactions. To facilitate these studies, gonococci were transformed with a hybrid shuttle vector containing the gfp gene from Aequoria victoria, encoding the green fluorescent protein (GFP), to produce intrinsically fluorescent bacteria. The model demonstrated that both pili and Opa proteins were important for both mediating gonococcal interactions with endometrial cells and inducing the secretion of pro‐inflammatory cytokines and chemokines. Pil+ gonococci showed high levels of adherence and invasion, regardless of Opa expression, which was associated with increased secretion of IL‐8 chemokine and reduced secretion of IL‐6 cytokine. Gonococcal challenge also caused increased secretion of TNF‐α cytokine, but this did not correlate with expression of pili or Opa, suggesting that release of components from non‐adherent bacteria may be involved in TNF‐α induction. Thus, the use of cultured primary endometrial cells, together with gonococci expressing green fluorescent protein, has the potential to extend significantly our knowledge, at the molecular level, of the role of this important human pathogen in the immunobiology of pelvic inflammatory disease.

Interaction of Neisseria meningitidis with Human Meningeal Cells Induces the Secretion of a Distinct Group of Chemotactic, Proinflammatory, and Growth-Factor Cytokines

ABSTRACT The interactions of Neisseria meningitidis with cells of the leptomeninges are pivotal events in the progression of bacterial leptomeningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the role of the leptomeninges in the inflammatory response. Following challenge with meningococci, meningioma cells secreted specifically the proinflammatory cytokine interleukin-6 (IL-6), the CXC chemokine IL-8, the CC chemokines monocyte chemoattractant protein 1 (MCP-1) and regulated-upon-activation, normal-T-cell expressed and secreted protein (RANTES), and the cytokine growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). A temporal pattern of cytokine production was observed, with early secretion of IL-6, IL-8, and MCP-1 followed by later increases in RANTES and GM-CSF levels. IL-6 was induced equally by the interactions of piliated and nonpiliated meningococci, whereas lipopolysaccharide (LPS) had a minimal effect, suggesting that other, possibly secreted, bacterial components were responsible. Induction of IL-8 and MCP-1 also did not require adherence of bacteria to meningeal cells, but LPS was implicated. In contrast, efficient stimulation of RANTES by intact meningococci required pilus-mediated adherence, which served to deliver increased local concentrations of LPS onto the surface of meningeal cells. Secretion of GM-CSF was induced by pilus-mediated interactions but did not involve LPS. In addition, capsule expression had a specific inhibitory effect on GM-CSF secretion, which was not observed with IL-6, IL-8, MCP-1, or RANTES. Thus, the data demonstrate that cells of the leptomeninges are not inert but are active participants in the innate host response during leptomeningitis and that there is a complex relationship between expression of meningococcal components and cytokine induction.

Immunization with Recombinant Opc Outer Membrane Protein from Neisseria meningitidis: Influence of Sequence Variation and Levels of Expression on the Bactericidal Immune Response against Meningococci

ABSTRACT The opc gene from Neisseria meningitidiswas cloned into the pRSETA vector, and recombinant protein was expressed at high levels in Escherichia coli. The protein was readily purified by affinity chromatography and used for immunization with conventional Al(OH)3 adjuvant or after incorporation into liposomes and Zwittergent micelles. The resulting sera were analyzed for their ability to recognize purified recombinant protein and “native” protein in an enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Immunization with Al(OH)3 induced high levels of antibodies which reacted with the purified protein but did not recognize whole cells. In contrast, liposomes and micelles induced antibodies which reacted with the native protein in whole cells. The addition of monophosphoryl lipid A (MPLA) to either liposomes or micelle preparations increased the magnitude of the immune response and induced a wider range of immunoglobulin subclasses. This was associated with the ability of the sera to induce complement-mediated killing of the homologous strain. The most effective bactericidal activity was observed with Opc protein incorporated into liposomes containing MPLA. The magnitude of the bactericidal effect was strongly influenced by the level of expression of the Opc protein and was abolished by limited variation in the sequence of the protein expressed by heterologous strains.